Apoptosis, or programmed cell death, is a conserved process that is mediated by the activation of a series of cysteine aspartyl-specific proteases termed caspases. Apoptosis plays an important role in a wide variety of normal cellular processes including fetal development, tissue homeostasis, and maintenance of the immune system (1). However, abnormal apoptosis can be involved with diseases such as ischemia-reperfusion injury (stroke and myocardial infarction), cardiomyopathy, neurodegeneration (Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, ALS), sepsis, Type I diabetes, fulminant liver disease, and allograft rejection (2,3). The beneficial effect of many drugs, especially antitumor drugs, can be attributed to their activation of the apoptotic process (26-31).
There are two different classes of caspases involved in apoptosis, the initiator caspases and the executioner caspases (5). The initiator caspases, which include caspase-6, -8, -9, and -10, are located at the top of the signaling cascade; their primary function is to activate the executioner caspases, caspase-2, -3, and -7. The executioner caspases are responsible for the physiological (e.g., cleavage of the DNA repair enzyme PARP-1, nuclear laminins, and cytoskeleton proteins) and morphological changes (DNA strand breaks, nuclear membrane damage, membrane blebbing) that occur in apoptosis (2). A third class of caspases, caspases-1, -4, -5, and -13, are involved in cytokine maturation and are not believed to play an active role in apoptosis.
Consequently, drugs targeting caspase-3 and caspase-7 have been important areas of pharmaceutical research. Most inhibitors of caspase-3 and caspase-7 are small peptides that inhibit caspase-3/7 by interacting either reversibly or irreversibly with cysteine-163 in the active site of the enzyme (6-13). However, peptide-based inhibitors typically have low bioavailability and are not effective in preventing apoptosis in vivo.
Ekici et al. described aza-peptide Michael Acceptors as inhibitors for cysteine proteases, including aza-Asp derivatives that were specific for caspases (40). A potential problems of peptide-based caspase inhibitors is their poor metabolic stability and poor cell penetration (12).
It was previously reported that isatin sulfonamides are potent and selective non-peptide-based inhibitors of the executioner caspases, caspase-3 and -7 (16). One compound, (S)-(+)-5-[1-(2-methoxymethyl-pyrrolidine)sulfonyl]isatin, 1 (FIG. 1) has been shown to reduce tissue damage in an isolated rabbit heart model of ischemic injury (14,15). Additional structure-activity relationship studies have revealed that replacement of the 2-methoxymethyl group with a phenoxymethyl moiety and the introduction of an alkyl group on the isatin nitrogen group results in improved potency for inhibiting caspase-3 activity (2) (FIG. 1) (16). An additional improvement in potency was also reported when the pyrrolidine ring of 3 (FIG. 1) was replaced with an azetidine ring to give compound 4 (FIG. 1) (16).
Positron emission tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are in vivo imaging techniques that measure changes in tissue and cellular function at the molecular level. Most agents used for imaging apoptosis in vivo are based on detection of Annexin V (32) and propidium iodide exclusion test (33), is required to discriminate between apoptosis and necrosis in vitro. Although such tests are routinely used to distinguish apoptosis from necrosis using ex vivo techniques such as flow cytometry, they cannot be applied to in vivo techniques such as PET and SPECT due to the short half-life radionuclides used.
A previous study reported the synthesis and carbon-11 radiolabeling of an isatin analog having a modest potency for inhibiting caspase-3 (38). However, no in vivo data were reported in this meeting abstract, and the selectivity of this compound for caspase-3 versus other caspases was not mentioned.
A potential disadvantage of known isatin analogues described as caspase inhibitors is that they are reversible inhibitors of caspase-3/7 since they form a thio-hemiketal with Cys-163 in the active site of activated caspase-3/7 (FIG. 14). Because current isatin analogues are predicted to be reversible inhibitors of activated caspase-3/7, they provide only temporary inactivation of the enzyme.